Electrofusion microelectrode

ABSTRACT

The present invention is directed to an electrofusion microelectrode used in the alignment, manipulation, fusion, or electroporation of cells. This device is particularly useful for transplantation of cells and cellular components.

This is a continuation of U.S. patent application Ser. No. 11/123,528,filed May 6, 2005, which claims benefit of U.S. patent application Ser.No. 10/090,036, filed Feb. 28, 2002, now U.S. Pat. No. 7,101,703, whichclaims benefit of U.S. Provisional Patent Application Ser. No.60/274,378, filed Mar. 9, 2001, which are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Electrofusion and electroporation of cells involves application of anelectrical current to cells. In many instances, cells are aligned priorto applying a direct electrical current. Alignment may be done manually,for example, by aspiration or vacuum suction. Alignment may also beperformed by applying an alternate electrical current. When alignment isdone by applying alternate current, cell survival is drasticallyreduced. The present invention provides a tool having the dual capacityto manually align cells and deliver direct current to cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of one embodiment of the electrofusionmicroelectrode. In this embodiment, the first end of the tube is sealed.

FIG. 2 is an illustration of another embodiment of the electrofusionmicroelectrode. In this embodiment, the first end of the tube is open.

FIG. 3 is an illustration depicting the electrofusion microelectrodeconnected to a power source via an electrode clip. In this illustration,the microfilament protrudes from the tube at the distal end and is bentor looped. The electrode clip is clamped on the distal end of the tubeand also contacts the bent portion or loop of the conducting filament.Suction means via a pipette holder is also depicted in this figure andmay be connected to the distal end of the electrofusion microelectrode.

DESCRIPTION OF THE INVENTION

The present invention is directed to an electrofusion microelectrodewhich may be used in the alignment, manipulation, fusion orelectroporation of cells including the transplantation of cells andcellular components. The electrofusion microelectrode comprises a tubeencasing a filament which is an electric conductor. As used herein,“tube” is meant to encompass any hollow casing and may have any type ofgeometrical conformation. Thus, if desired, the walls of the tube may beangled. In a preferred embodiment, the tube is cylindrical. In an evenmore preferred embodiment, the tube is shaped as a holding pipette.

The tube as well as the conducting filament has both a medial and distalend. As used herein, the “medial end” of the tube or conducting filamentis the end which contacts the cells and/or cellular components. The“distal end” of the tube or conducting filament is furthest away fromthe cells and/or cellular components and nearer a direct current powersource.

The conducting filament may comprise any known conductor such as ametal, metal alloy or mixture of metals and/or metal alloys. Certaincarbon allotropes may also be used to form the conducting filament.Examples of metal conductors which may be used as a conducting filamentinclude but are not limited to, aluminum, copper, silver, gold,titanium, platinum, and tungsten. An example of a carbon allotrope whichmay be used as a conducting filament in the electrofusion microelectrodeis graphite. In a preferred embodiment, the metal filament is made oftungsten or tungsten alloy.

In one embodiment of the invention, one end of the filament is flattenedat the tip of one end of the tube and the tube is sealed at this end(medial end). In an alternative embodiment, the electrofusionmicroelectrode has an internal opening surrounding the filament. Theinternal opening is useful to allow aspiration or vacuum suctioning ofcells such as used with a standard holding pipette. In this embodiment,the end of the tube which is in contact with the cells or cellularcomponents, i.e., the medial end, is open. In FIG. 1, the first (medial)end of the tube where the first (medial) end of the filament protrudesis sealed. FIG. 2 shows an alternative embodiment where the first end ofthe tube is open.

The tube portion of the electrofusion microelectrode may be made of anynumber of materials such as glass, plastic, PVC, ceramic, metal, etc. Ina preferred embodiment, the tube portion is made of glass. In a morepreferred embodiment, the tube portion is made from a borosilicate glasscapillary tube, pulled and forged as a holding pipette.

The length and diameter of the electrofusion microelectrode may varyaccording to the type of cells and type of manipulation for which thetool is used. For example, when used for nuclear transplantation ofmammalian cells, a tube diameter in the range of from about 15 to about25 μm is useful. When used for mammalian cell fusion, a tube diameter inthe range of from about 60 to about 100 μm may be used. Thus, in oneembodiment, the outer diameter of the tube may be about 0.97 mm whilethe inner diameter of the tube may be about 0.69 mm. In this embodiment,the tube is quite thin walled, having a thickness of only about 0.28 mm.The diameter of the conducting filament may be anywhere in the range offrom about 7 to about 20 μm. The distal end of the conducing filament ispreferably thicker than the medial end so that connection to a powersource is conveniently achieved.

The length of the microelectrode can of course, vary. A length of about78 mm is convenient for most manipulations. Typically, there is a bendin the tube approximately 1 mm or so from the medial end.

With reference to FIG. 1, one embodiment of the invention is illustratedtherein and it will be seen to include an electrode main body and anelectrode tip. As illustrated in FIG. 1, a conductor filament extendsthroughout the tube (electrode main body). Both the tube and thefilament have a first (medial) and a second (distal) end. A first(medial) end of a filament protrudes through a first (medial) end of thetube and is flattened at the tip of the first (medial) end of the tube.In FIG. 1, the medial ends make up the electrode tip. A second (distal)end of the filament protrudes through a second (distal) end of the tubeand is configured to both allow the filament to remain relatively fixedwithin the tube and to allow connection to a power source. The medialend of the tube may be open, or closed (sealed).

There are many possible configurations for the distal, protruding end ofthe filament. In one embodiment, the distal end of the filament may bebent or looped towards the outer wall of the tube or wrapped around theouter wall of the tube in order to have the filament remain relativelyfixed within the tube. Conveniently, an electrode clip or the like maybe clamped around the tube as well as the looped, bent, or wrappedportion of the distal end of the filament. This embodiment of theinvention is depicted in FIG. 3.

In another embodiment of the invention, the medial end of the conductingfilament does not protrude from the medial end of the tube. The innerwalls of the tube are painted with a liquid form of an electricconductor from a place where the filament no longer extends to themedial end of the tube and the paint extends to the outside (lateral)edge of the medial end of the tube. In this embodiment, the distal endsof the filament and tube are as described above. Again, the medial endof the tube may be open or closed (sealed).

In yet another embodiment of the invention, rather than using aconducting filament, the inner portion of the tube is painted with aliquid form of an electric conductor. Examples include liquid aluminum,copper, silver, gold, titanium, platinum, tungsten, and alloys andmixtures thereof. Thus, at least a portion of the inner walls arepainted with a liquid electric conductor and the painted area extendscontinually from the medial end of the tube to the distal end of thetube. The medial end of the tube may be opened or sealed. The liquidconductor is also painted on at least a portion of the outer (lateral)edge of both the medial and distal ends of the tube. Further, a portionof the liquid conductor is applied to the outside wall of the tube atthe distal end so that connection to a direct current power source maybe achieved. For example, an electrode clip may be clamped to the tube,contacting that portion of the distal end of the outer wall of the tubewhich is painted with the liquid conductor.

The electrofusion microelectrode is preferably mounted on a tool holderwhere it can be controlled by a micromanipulator. Preferably, themicromanipulator is used under inverted microscopy. Examples ofmicromanipulators which may be used with the subject electrofusionmicroelectrode include but are not limited to, the MM188 and MM109manufactured by Narishigie Co., LTD, Tokyo, Japan. Preferably, theelectrofusion microelectrode is used as a set of two: the distal end ofthe conducting filament of one electrofusion microelectrode beingconnected to the positive terminal of a direct current power source, andthe distal end of the conducting filament of a second subjectelectrofusion microelectrode being connected to the negative terminal ofa direct current power source. The power source should be able todeliver at least 1 kilovolt per centimeter, direct current. Examples ofpower sources that may be used with the subject microelectrode includethe BTX Electro Cell Manipulator 200 or 2001 (BTX Inc., San Diego,Calif.).

The subject tool(s) may be used to perform the techniques ofelectrofusion/electroporation by manually aligning and/or ormicromanipulating the cells using microelectrode motion. Alternatively,if the medial end of the tool(s) is open, cells may be manually alignedusing aspiration or vacuum suction. Of course, a combination ofmicroelectrode motion and aspiration or suction may be used tomicromanipulate and/or align cells. After aligning cells, direct currentmay be applied via the subject microelectrode(s). Since cells arealigned manually, the use of alternate current for alignment is avoided,significantly improving cell survival. Since cell survival isdrastically improved, much lower cell numbers may be used in eachmanipulation.

The present invention therefore provides methods of manipulating cellsusing the subject electrofusion microelectrode. Such methods include forexample, cell transplantation, electrofusion of cells, electroporationof cells, and nuclear transplantation. Thus, the present inventionprovides a method of transplanting mammalian cells which comprisesmicromanipulating the cells with two electrofusion microelectrodes anddelivering a direct current to the manipulated cells. The subjectelectrofusion microelectrodes for use in the method of transplantationof mammalian cells, may have any of the alternate embodimentshereinbefore described.

The present invention also provides a method of electrofusion of cells.The method comprises aligning cells between two electrofusionmicroelectrodes and delivering a direct current to the aligned cells.Again, the subject electrofusion microelectrodes for use in the methodof electrofusion of cells, may have any of the alternate embodimentshereinbefore described.

Also provided by the present invention is a method of electroporation ofcells. The method comprises manipulating cells with two electrofusionmicroelectrodes and delivering a direct current to the manipulatedcells. The subject electrofusion microelectrodes for use in the methodof electroporation may have any of the alternate embodimentshereinbefore described.

A method of nuclear transplantation is also provided by the presentinvention. The method comprises removing a nucleus from a first oocyteand transplanting the nucleus into the perivitelline space of a second,previously enucleated oocyte, and then integrating the transplantednucleus of the first oocyte with the cytoplasm of the second oocyte.Transplantation and integration is performed using the subjectelectrofusion microelectrodes and integration is achieved by deliveringa direct current to the nucleus and cytoplasm. The subject electrofusionmicroelectrodes for use in the method of nuclear transplantation mayhave any of the alternate embodiments hereinbefore described.

EXAMPLE I

A capillary tube, 78 mm in length, and having an outer diameter of 0.97mm and an inner diameter of 0.69 mm (Drummond Scientific, Boomall, Pa.),is pulled on a horizontal microelectrode puller (micropuller) (CampdenInc., LTD., London) approximately 60 to 100 μm at a location of 10-15 mmfrom one end (medial end). The tube is cut and fine polished on amicroforge (Narishige Co., LTD, Tokyo, Japan) to obtain a final outerdiameter of 60 μm and an inner diameter of 20 μm. A platinum filamenthaving a thickness of about 20 to 40 μm (available from a fine jeweler)is inserted into the distal end of the pipette under a sterilemicroscope with a magnification of 6-15× or a magnifying lens of atleast 6×. The medial end of the conducting filament is placed flushagainst the tip of the medial end of the pipette. The distal end of thefilament is of a length longer than the pipette so that that it exitsthe distal end of the pipette by a length of at least 10 mm. Thisportion of the filament which exits the distal end of the pipette isbent towards the outside wall of the distal end of the pipette, making abend or a loop to secure the filament in place within the pipette and toallow connection to a power source by means of an electrode clip. Anelectrode clip may be attached to the tube, ensuring that contact withthe protruding portion of the distal end of the filament is made (FIG.3). The electrode clip may be connected to a direct current power sourcesuch as the BTX Electro Cell Manipulator 200 or 2001 (San Diego,Calif.).

EXAMPLE II Nuclear Transplantation for Immature Mammalian Oocytes

Germinal vesicle (GV) stage oocytes are retrieved by puncturingfollicles of unstimulated ovaries of B6D2F1 female mice. A karyoplast isthen removed by micromanipulation using one or more of the subjectelectrofusion microelectrodes in a medium supplemented with cytochalasinB. One karyoplast is subsequently introduced into the perivitellinespace of a previously enucleated immature oocyte. Each grafted oocyte isthen positioned between two of the subject electrofusion microelectrodesand exposed to a single or double 1.0 kV/cm, 50-99 μm direct currentfusion pulse(s). Thirty to 60 minutes later, the oocytes are examinedfor sign of fusion. The restored oocytes are then placed in culture andassessed for maturation. Oocytes which have extruded a first polar bodymay be fixed and stained with Giemsa for chromosome analysis. Ascontrols, approximately one third of oocytes are not subjected to anymanipulation, but are merely cultured in the same media and exposed tosame reagents.

EXAMPLE III Germinal Vesicle Transplantation

Germinal vesicle (GV) stage oocytes are retrieved by puncturingfollicles of unstimulated ovaries of B6D2F1 female mice. Metaphase II(MII) oocytes are collected 15 hours after hCG injection of PMSGstimulated females. Karyoplasts are then removed from GV oocytes usingone or more subject electrofusion microelectrodes, in a mediumsupplemented with cytochalsin B. MII oocytes are enucleated by removingthe “hub” area where the metaphase spindle is located, together with thefirst polar body using one or more of the subject electrofusionmicroelectrodes. A GV karyoplast is subsequently introduced into theperivitelline space of either a previously enucleated immature (GV) or amature (MII) oocyte. Each of these manipulated oocytes is thenpositioned between two of the subject electrofusion microelectrodes andexposed to a single or double 1.0 kV/cm, 50-99 μm direct current fusionpulse(s) for electrofusion. The oocytes that show signs of fusion 30 to60 minutes later are then placed in culture for 12 hours, to allownuclear maturation. Oocytes which extrude the first polar body may befixed and stained with Giemsa for chromosome analysis.

1. An electrofusion microelectrode which comprises a tube having walls painted with a liquid electric conductor and wherein the painted electric conductor extends continually from a first (medial) end of the tube to a second (distal) end of the tube, wherein the distal end of the tube is connectable to a direct current power source.
 2. The electrofusion microelectrode of claim 1 wherein the tube is shaped as a holding pipette.
 3. The electrofusion microelectrode of claim 1 wherein the first (medial) end of the tube is sealed.
 4. The electrofusion microelectrode of claim 1 wherein the first (medial) end of the tube is open.
 5. The electrofusion microelectrode of claim 1 wherein the tube is made of plastic, PVC, ceramic, or metal.
 6. The electrofusion microelectrode of claim 1 wherein the tube is made of glass.
 7. The electrofusion microelectrode of claim 1 wherein the tube is bent.
 8. The electrofusion microelectrode of claim 1 wherein the second (distal) end of the tube is connectable to a vacuum or hand held aspirator.
 9. The electrofusion microelectrode of claim 8 wherein the hand held aspirator is a pipette holder.
 10. The electrofusion microelectrode of claim 1 further comprising: a tool holder on which the electrofusion microelectrode is mounted.
 11. The electrofusion microelectrode of claim 10 further comprising: a micromanipulator which controls the tool holder.
 12. The electrofusion microelectrode of claim 1 wherein the liquid electrical conductor is selected from the group consisting of liquid aluminum, copper, silver, gold, titanium, platinum, tungsten, alloys, and mixtures thereof. 